ATP citrate lyase knockdown impacts cancer stem cells in vitro.

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer stemness in a broad range of genetically diverse cell types.

Is the mass screening for neuroblastoma ineffective?

Though a recent study (Schilling et al. 2002) concluded that the mass screening for neuroblastoma targeting children age 12 months was ineffective, we pointed out several serious problems and reestimated its effectiveness using their data. They employed the subjects in the "control area" as controls, not the "non-participants" whose biases are fewer because their area is the same as that of the participants. The incidence of neuroblastoma among the subjects in the "control area" was about 25% smaller than that of the "non-participants". This leads to underestimation of the effectiveness of the mass screening. They combined false negatives with true positives to calculate the incidence of the "screened group". But since many spontaneous regression cases are included in the true positives, this method inflates the incidence of the "screened group", leading to underestimation of the effectiveness of the mass screening. When the false negatives are compared with the non-participants, the incidence of the cases in stage 4 among the latter is about 40% of that of the former, and the mortality is less than two-thirds. The percentage of spontaneous regression cases among the true positives is estimated to be about 40%. These results are better than those of the Japanese screening programs (targeting infants age 6 months), supporting the effectiveness of mass screening for neuroblastoma.

Contribution of HPLC mass screening for neuroblastoma to a decrease in mortality.

The aim of this study is the estimation of the contribution of HPLC mass screening for neuroblastoma to the decrease in deaths due to this disease. The mortality rates of malignant neoplasms of the adrenal glands (ICD 9, 1940; ICD 10, C74; virtually all the cases of these codes are neuroblastoma during childhood) at 1-4 years of age in cohorts born in 1979-1984, 1985-1988, and 1989-1992 in the whole of Japan were calculated, using data obtained from the Ministry of Health and Welfare. The numbers of infants screened by HPLC in the cohorts were estimated through the reports of the Ministry of Health and Welfare and the database of the Japanese Society for Mass-screening. The mortality of the cohort born in 1989-1992, in which 77.8% of the live births were screened by HPLC, was 1.73 per 100,000 live births. This is about half of that (3.26) of the cohort born in 1979-1984, in which few infants were screened. On the assumption that cases of the 1985-1988 and 1989-1992 cohorts died according to the mortality rate of the 1979-1984 cohort, the expected numbers of deaths were estimated; that for the 1985-1988 cohort was 178.51 (of them, that for the infants screened by HPLC was 39.65), and that for the 1989-1992 cohort was 159.78 (of them, that for the infants screened by HPLC was 124.33). The observed numbers of deaths were 145 and 85, respectively. Assuming that non-HPLC methods have no effects and using 2 unknown quantities x (contribution of HPLC) and y (other factors), simultaneous equations (1) 178.51 - 39.65x - 178.51y = 145 and (2) 159.78 - 124.33x - 159.78y = 85 were made. Solving them, x = 0.5041 and y = 0.0757 were obtained. In conclusion HPLC screening targeting infants aged 6 months reduces death of adrenal neuroblastomas at 1-4 years of age by about 50%.

Expression of Ca(2+)-activated K(+) channels, SK3, in the interstitial cells of Cajal in the gastrointestinal tract.

A role for small-conductance Ca(2+)-activated K(+) (SK) channels on spontaneous motility of the gastrointestinal tract has been suggested. Although four subtypes of SK channels were identified in mammalian tissues, the subtypes of SK channel expressed in the gastrointestinal tract are still unknown. In this study, we investigated the expression and localization of SK channels in the gastrointestinal tract. RT-PCR analysis shows expression of SK3 and SK4 mRNA, but not SK1 or SK2 mRNA, in the rat intestine. SK3 immunoreactivity was detected in the myenteric plexus and muscular layers of the stomach, ileum, and colon. SK3-immunoreactive cells were stained with antibody for c-kit, a marker for the interstitial cells of Cajal (ICC), but not with that for glial fibrillary acidic protein in the ileum and stomach. Immunoelectron microscopic analysis indicates that SK3 channels are localized on processes of ICC that are located close to the myenteric plexus between the longitudinal and circular muscle layers and within the muscular layers. Because ICC have been identified as pacemaker cells and are known to play a major role in generating the regular motility of the gastrointestinal tract, these results suggest that SK3 channels, which are expressed specifically in ICC, play an important role in generating a rhythmic pacemaker current in the gastrointestinal tract.

Voided urine cytology of papillary renal cell carcinoma and renal calculus: report of a case with emphasis on the importance of cytologic screening in high-risk individuals.

BACKGROUND: Preoperative diagnosis of cases of renal calculus complicated with papillary renal cell carcinoma (RCC) by image analysis is usually difficult. CASE: A 50-year-old man who had a past history of renal calculus suffered from macrohematuria and abdominal pain for one month was admitted to our hospital. Ultrasonographic examination revealed a 4-cm tumor shadow in the right kidney; it was hypovascular in arteriography. Papillary cell clusters with abundant cytoplasm were found by the cytologic examination of voided urine. Their nuclei were oval and situated eccentrically in the cytoplasm. The nuclear/cytoplasmic ratio was increased. Fine, granular chromatin was distributed evenly, and the nuclear membrane was thin and nearly smooth. Several small nucleoli were evident. All these findings were indicative of a diagnosis of papillary RCC. Histology of nephrectomy specimens confirmed the diagnosis. CONCLUSION: Voided urine cytology can be useful for screening and follow-up of patients with papillary RCC.

Cell surface glypicans are low-affinity endostatin receptors.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Lymphoproliferative disorders in renal transplant patients in Japan.

Post-transplantation lymphoproliferative disorders (PT-LPD) are characterized by a clinically and morphologically heterogeneous group of lymphoid proliferation occurring after organ or bone marrow transplantation. The immunodeficient state provides a basis for lymphomagenesis probably through activation of oncogenic viruses. Twenty-four patients in whom PT-LPD developed after renal transplantation in Japan were analyzed. They received hemodialysis for 4 to 226 (median 13) months before transplantation. In situ hybridization was performed to detect Epstein-Barr virus (EBV). Polymerase chain reaction and Southern hybridization with primers in the tax and pol regions of human T-cell leukemia virus type I (HTLV-1) were performed on DNA extracted from paraffin-embedded specimens. Immunohistochemical analysis revealed that 12 cases were B-cell type, 10 cases (42%) T-cell type and 2 NK-cell type. Five of the T-cell cases were classified as adult T-cell lymphoma with proven HTLV-1 genome in the tumor and seropositivity for the virus. These cases were classified as adult T-cell lymphoma (ALT). More than 80% of B-cell, 30% of T-cell and both NK/T-cell lymphomas were EBV-positive. Co-infection of EBV and HTLV-1 was found in 2 cases with ATL. These findings showed that ATL is common among Japanese renal transplant patients, which might be due to transmission of HTLV-1 via blood transfusion during hemodialysis.

The use of an osseointegrated implant for orthodontic anchorage to a Class II Div 1 malocclusion.

This case report describes the use of an osseointegrated implant to maximize anchorage in a 24-year-old female orthodontic patient with an Angle Class II, Division 1 malocclusion. Preadjusted edgewise appliance therapy was performed by extraction of only the maxillary first premolars. The osseointegrated implant was placed in the median-sagittal region of the hard palate for maximum orthodontic anchorage and connected to maxillary first molar bands via a transpalatal arch. Total treatment time was 2 years and 8 months. Cephalometric superimposition revealed the achievement of maximum molar anchorage in the maxilla, resulting in satisfactory occlusal and facial improvements. Histological analysis of the implant-bone interface demonstrated that the fixture was successfully osseointegrated. In conclusion, the osseointegrated implant placed in the median-sagittal palate was shown to be an effective orthodontic system that can be used clinically as a rigid intraoral anchorage.

Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA.

Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

A case of breast cancer diagnosed by inguinal lymph node metastasis.

We describe a case of a 58-year-old woman with right inguinal lymph node swelling and a T1 tumor in the right breast. She was referred with an 18-month history of the former complaint and a six-month history of the latter. Excisional biopsy of the inguinal lymph node revealed breast cancer metastasis. Radiographical examination showed no metastases to the lungs, liver or bone. Modified radical mastectomy was performed. Histological examination revealed solid tubular carcinoma, PT2, PM (axillary lymph node metastases 4/16), stage IV. Estrogen and progesterone receptors were negative. Three cycles of postoperative cyclophosphamide, adriamycin and 5-fluorouracil (CAF) chemotherapy were given, and the right inguinal area was irradiated with 40 Gy. The patient complained of swelling in both legs three years after surgery. Computed tomography revealed marked lymph node swellings in the pelvic cavity. She died six months later. Inguinal lymph node metastasis from breast cancer is very rare, although distant lymph node metastasis in the cervix occurs frequently. This case should help clarify how breast cancer metastasizes to distant lymph nodes.

A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD), an autosomal-dominant human bone disease, is thought to be caused by heterozygous mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1). To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, CCDalphaA376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We show that signal transducers of transforming growth factor beta superfamily receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance the transactivation ability of this factor. The truncated RUNX2 protein failed to interact with and respond to Smads and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts on stimulation by bone morphogenetic protein. Therefore, the pathogenesis of CCD may be related to the impaired Smad signaling of transforming growth factor beta/bone morphogenetic protein pathways that target the activity of RUNX2 during bone formation.

Schnurri interacts with Mad in a Dpp-dependent manner.

BACKGROUND: Decapentaplegic (Dpp) is a member of the transforming growth factor-beta superfamily. Dpp governs various developmental processes in Drosophila through the transcriptional regulation of a variety of genes. Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad was shown to bind to the Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. RESULTS: We characterized the biochemical functions of Shn. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Finally, we found that Shn interacts with Mad in a Dpp-dependent manner. CONCLUSIONS: The present results argue that Shn may act as a nuclear component of the Dpp signalling pathway through direct interaction with Mad.

Multiple inflammatory pseudotumors mimicking liver metastasis from colon cancer: report of a case.

A 54-year-old man underwent an operation for colon cancer histologically diagnosed as moderately differentiated adenocarcinoma with clinical staging of Dukes C. He was prescribed carmofur for adjuvant chemotherapy. A follow-up computed tomography scan done 6 months later revealed two new low-density areas in the liver. A diagnosis of metastatic adenocarcinoma from the previous colon cancer was presumed, based on the patient's history and radiological findings, and resection of the affected area of liver was performed. Histological examination of these tumors revealed that they were inflammatory pseudotumors (IPT). The patient had an excellent postoperative course and has shown no further signs of recurrence in the 3 years since his last operation. IPT of the liver is a rare disease, for which no methods of diagnosis and treatment have been established, since it is difficult to distinguish IPT from hepatocellular carcinoma or metastatic carcinoma. We describe this case with a review of the 101 cases of IPT documented in the Japanese literature, in the hope that it will contribute to the diagnosis and treatment of this unusual disease entity.

Catheterized urine cytology of mucinous carcinoma arising in the renal pelvis. A case report.

BACKGROUND: Primary mucinous carcinoma of the renal pelvis is a rare tumor; therefore, criteria for cytologic diagnosis of this tumor have not been established. CASE: An 81-year-old woman suffered from macrohematuria for six months and was found to have a tumor in the right kidney by radiographic examination. Catheterized urine obtained from the right renal ureter was viscous and contained spherical clusters of cells with occasionally vacuolated, lacy and basophilic cytoplasm. In the small to medium-sized nuclei, chromatin was coarse and granular, and the nuclear membrane was thin and nearly smooth. Large nucleoli were evident in some of the nuclei. These findings were consistent with adenocarcinoma possibly of mucinous type. CONCLUSION: Preoperative diagnosis of mucinous carcinoma is possible by cytologic findings of catheterized urine together with clinical data.

[Changes in MR imaging appearance of breast cancer after intra-arterial infusion chemotherapy].

The purpose of this study was to evaluate whether the characteristic change in breast cancer related to chemotherapeutic response (CR) and the effect of invasion and toxicity in the skin and pectoralis muscle exist on MR imaging after intra-arterial infusion chemotherapy. A total of 11 patients with histologically proven breast cancer underwent MR study before and after chemotherapy. Changes in images and the dynamic curve-after-chemotherapy were evaluated, including time to maximum signal intensity (SI) and the early phase enhance ratio (EPER) in the tumor. In the tumor, changes in the dynamic curve, time to maximum SI, EPER and necrosis did not correlate with CR, but change in SI on T2-weighted images was suggested to do so. Changes in the dynamic curve and images in the pectoralis muscle and in images on the skin were suggested to correlate with CR. In addition, images changed for the worse in many cases of invasion and toxicity in the pectoralis muscle and in some cases of invasion in the skin. In conclusion, tumors had fewer imaging changes correlating with CR after intra-arterial infusion chemotherapy. Changes for the worse in images of the pectoralis muscle and skin may be useful for the evaluation of invasion.

c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads.

Smads are intracellular signaling mediators of the transforming growth factor-beta (TGF-beta) superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-beta. We identified c-Ski as a Smad2 interacting protein. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-beta-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-beta. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-beta signaling.

Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter.

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha and beta subunits. The alpha subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-beta/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2alphaC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Calpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-beta/BMP signaling.

E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins.

Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily. Smads bind to DNA and act as transcriptional regulators. Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction. Smads also associate with transcriptional coactivators such as p300 and CBP. E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition. Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction. One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region. p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner. E1A inhibits the interaction of Smad3 with a p300 mutant that contains SID but lacks the E1A binding domain. We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby E1A antagonizes TGF-beta signaling.